1. Field of the Invention
The invention relates to a novel buffer formulation for fast separation, purification and highly efficient recovery of long- and/or short-chain nucleic acids.
2. Discussion of the Background
A multiplicity of commercially-available kits exist today for the purification and recovery of specific DNA fragments.
All of these methods are based on a method for the preparative and analytical purification of DNA fragments from agarose gels developed and described for the first time by Vogelstein and Gillespie (Proc. Natl. Acad. Sci. USA, 1979, 76, 615-619). The method combines the break-up (in a saturated solution of a chaotropic salt (NaI)) of the agarose containing the bands of the DNA to be isolated with binding of the DNA to glass particles. The DNA fixed to the glass particles is then washed with a wash solution (20 mM Tris HCl [pH 7.2]; 200 mM NaCl; 2 mM EDTA; 50% v/v ethanol) and finally separated from the support particles.
The physico-chemical principle of the systems for isolating nucleic acids on the basis of the binding of nucleic acids to the surfaces of mineral supports, adopted and commercially available today according to the background art, should thereby consist in the disruption of superordinate structures of the aqueous medium, through which the nucleic acids on the surface of mineral materials, in particular of glass or silica particles, adsorb. The disruption of the superordinate structures of the aqueous medium is thereby always carried out in the presence of chaotropic ions and is almost quantitative at high concentrations of these. On this physico-chemical basis described, all commercially available systems for the isolation of nucleic acids contain buffer compositions with higher ion strengths of chaotropic salts, for the binding of nucleic acids to a nucleic acid-binding solid phase.
All the methods described for the isolation of nucleic acids by the binding of nucleic acids to mineral solid phases by the use of chaotropic salt solutions have in common the fact that for the binding of the nucleic acids to the support materials used, high concentrations must be adopted. At the same time, chaotropic salts (e.g. guanidine thiocyanate, guanidine hydrochloride, sodium perchlorate or sodium iodide) are highly toxic potent substances. The buffer systems with very high ion strengths being used often effect a diversion of salt contaminations, which can prove problematic for a string of downstream applications. What is more, in association with chaotropic buffers there exists a considerable health risk (in particular in long-term use) as well as a considerable environmental effect through quantities of toxic substances dumped into effluent water.
Interestingly, it turns out that all systems which are commercially available worldwide for isolating nucleic acids on the basis of binding of nucleic acids to mineral support materials (magnetic particles, membranes, carrier suspensions et al.) work, in principle, according to the method described. Since the first description by Vogelstein and Gillespie, the bound nucleic acids have always been washed with alcohol or salt solutions containing acetone. The wash steps are essential components of the extraction protocols and, alongside the removal of bound, undesired, inhibiting substances, always also serve in the necessary removal of the salts necessary for the binding of the nucleic acids.
In WO 01/62976 A1, a description is disclosed which comprises the purification of nucleic acids from various reaction assays upon addition of different alcohols, their subsequent precipitation on special solid phases (membranes with specific physical characteristics), wash steps with alcoholic buffers and the final elution of the nucleic acids by means of water.
U.S. Pat. No. 5,405,951 A and EP 0512767 A1 likewise describe the isolation of nucleic acids by incubation of the sample containing nucleic acids with an alcohol, and the subsequent incubation of the sample with a mineral material. The elution of the nucleic acids is carried out upon the addition of water heated to 60° C.
In DE 10253351 A1, it is disclosed that the purification and recovery of nucleic acids is carried out by adjusting the solution containing nucleic acid with additives in such a way that it contains monovalent and multivalent cations as well as an alcohol, brings them afterwards into contact with the solid phase, subsequently washes the support and releases the nucleic acid from the solid phase. Ammonium chloride, sodium chloride and/or calcium chloride are used as monovalent salt components, and magnesium chloride, calcium chloride, zinc chloride and/or manganese chloride are used as multivalent salt components.
It is disclosed that precisely the combination of a monovalent and of a multivalent salt leads to nucleic acids adsorbing on solid phases, wherein the ion strengths necessary for this must only be very small. This has the advantage, if applicable, that wash steps which were always necessary until now are no longer required, and so the methods for isolating nucleic acids can be clearly shortened and simplified.
At the same time, it emerges that on use of buffer combinations given in DE 10253351 A1 (e.g. magnesium chloride/calcium chloride), the purification and recovery of DNA fragments from PCR reaction mixtures certainly takes place at a high recovery rate, but unfortunately no selective removal of undesired PCT by-products (e.g. primer-dimers) is possible.
For this reason, only the general possibility of the recovery of DNA fragments can be demonstrated, but not their efficient purification. The cause of this observation thereby seems to possibly be the multivalent cation used. If, however, the multivalent cation is removed from the buffer mixture, then the recovery of nucleic acids with the buffers described is no longer possible on the basis of the very small ion strengths. However, the use of buffers with only very small ion strengths was precisely the inventive purpose of the patent specification.
WO/34463 A1 describes a method for isolating nucleic acids, in which the nucleic acids are bound to a solid phase, wherein the conveyance of the binding is carried out by binding buffers on the basis of so-called antichaotropic salts and an alcoholic component. The bound nucleic acids are washed with wash buffers known in themselves and finally eluted by the addition of a low-salt buffer. The ion strengths of the so-called antichaotropic salts come to at least 0.1M-10M. The so-called antichaotropic salts for the binding of nucleic acids to a solid phase are, without exception, chlorides.
WO 89/08257 A1 indicates that citrates belong to the group of antichaotropic salts. The property characterized in WO 89/08257 A1 is to be understood in the context of the immobilization of proteins and has no relationship at all to nucleic acids or methods for the isolation and purification of nucleic acids.